Natural attenuation and bioremediation of Prestige fuel oil along the Atlantic coast of Galicia (Spain)

J.R. Gallego a'*, E. Gonzalez-Rojas b , A.I. Pelaez b , J. Sanchez b , M.J. Garcia-Martinez c , J.E. Ortiz c , T. Torres c , J.F. Llamas c

a Dpto. Exp. y Prospeccion de Minas e Institute* de Biotecnologia de Asturias (IUBA), Universidad de Oviedo. CIGonzalo G, SIN - 33600 Mieres, Asturias, Spain

Area de Microbiologia, Dpto. Biologia Funcional e Instituto de Biotecnologia de Asturias (IUBA), Universidad de Oviedo. CUulian Claveria, SIN - 33006 Oviedo, Asturias, Spain

0 Laboratorio de Estratigrafia Biomolecular, ETSMinas, Universidad Politecnica de Madrid, CIRios Rosas, 21- 28003 Madrid, Spain

Abstract

Heavy fuel oil spilled from the oil tanker Prestige in November 2002 affected hundreds of km of Spanish shoreline. We carried out a two year study at two highly contaminated sites in order to monitor natural at tenuation of the residues coat­ing shore rocks and to test the effectiveness of bioremediation with an oleophilic fertilizer (S200). The methodology included an innovative approach for oil load calculation (based on image analysis techniques), the analysis of the fate of hydrocarbons by means of chemical biomarkers and different microbiological techniques for isolating and examining hydrocarbon degraders.

Considerable hydrocarbon depletion from shorelines (^100% for light-medium linear alkanes and ^ 3 5 % for aromatics such as pyrene) was observed within the first months after the spill. However, this natural at tenuation effect dramatically slowed down in the following year, al though partial enhancement (especially for the aromatic fraction) was attained as a result of the application of S200 to stimulate indigenous microorganisms. In addition, one of the main outcomes of this work concerns the remarkable hydrocarbon depletion achieved at points where fresh water flowed through the shore rocks. The study of samples collected where this natural attenuation phenomenon took place made it possible to isolate hetero­trophic bacteria and fungi that are likely to cooperate with cyanobacteria in hydrocarbon biodegradation. The overall results strongly advocate the implementation of new bioremediation approaches, including alternatives such as the use of natural fresh water to irrigate polluted areas.

1. Introduction

I n N o v e m b e r 2002 , t he oil t a n k e r Prestige spi l led a n as yet u n d e t e r m i n e d a m o u n t of h e a v y fuel n o . 6 t h a t e n d e d u p c o a t i n g h u n d r e d s of k m of t h e A t l a n ­t ic a n d C a n t a b r i a n c o a s t s in S p a i n (F ig . 1). A t m a n y sites, m e c h a n i c a l r e m o v a l of t h e fuel w a s

u n d e r t a k e n m a n u a l l y o n s a n d y b e a c h e s a n d us ing h o t p r e s su r i zed w a t e r w a s h i n g w h e r e v e r t h e fuel oil w a s firmly a d h e r i n g t o t h e sur face of la rge b o u l ­de r s a n d m a n m a d e s t r u c t u r e s . Al l these sys t ems h a d l imi ted effectiveness; h o w e v e r , a t sites subject t o specia l p r o t e c t i o n for e n v i r o n m e n t a l r e a s o n s , a n d a l o n g shore l ines w h e r e t h e a v e r a g e g r a i n size w a s p e b b l e o r c o b b l e size, these t e c h n i q u e s w e r e n o t a t all a d e q u a t e . T h e r e f o r e , a l t e rna t ives b a s e d

o n b i o r e m e d i a t i o n a n d / o r m o n i t o r i n g of n a t u r a l a t t e n u a t i o n w e r e r e c o m m e n d e d for ce r t a in selected l o c a t i o n s .

After a n ini t ia l p r o c e s s of emuls i f ica t ion , d i sper ­s ion a n d p a r t i a l d i s s o l u t i o n whi le f loat ing o n m a r i n e w a t e r ( D a l i n g a n d S t r o m , 1999; M o l d e s t a d t e t al . , 2004) , t h e Prestige fuel oil r e a c h e d s h o r e , w h e r e it u n d e r w e n t a va r i e ty of n a t u r a l a t t e n u a t i o n (wea the r ­ing) p roces ses i n c l u d i n g e v a p o r a t i o n , p h o t o c h e m i c a l

o x i d a t i o n a n d b i o d e g r a d a t i o n ( Z h u et al . , 2001) . T h u s , t he s t u d y of specific a r ea s of t h e p o l l u t e d sites w h e r e e n v i r o n m e n t a l p a r a m e t e r s ( n u t r i e n t s , h u m i d ­ity, o x y g e n a t i o n , etc.) a r e o p t i m a l for n a t u r a l a t t en ­u a t i o n c a n p r o v i d e useful i n f o r m a t i o n for t he des ign of e n h a n c e d b i o r e m e d i a t i o n t e c h n i q u e s a n d , conse ­quen t ly , w a s o n e of m a i n scopes of th i s w o r k . H o w ­ever , t he h e t e r o g e n e i t y of t he s e d i m e n t s i nvo lved a n d the v a r i a b l e spa t ia l d i s t r i b u t i o n of b o t h fuel a n d h y d r o c a r b o n d e g r a d e r s fu r the r e n d o r s e t h e n e e d t o p e r f o r m p r o p e r n a t u r a l a t t e n u a t i o n m o n i t o r i n g a n d p i lo t scale b i o r e m e d i a t i o n e x p e r i m e n t s p r i o r t o full scale i m p l e m e n t a t i o n of a n y b io log ica l t r e a t m e n t ( X u a n d O b b a r d , 2004) . H e n c e , t he very first p r o b ­lems t o b e a d d r e s s e d in these t ypes of s tud ies re la te t o def in ing p a r a m e t e r s such as t h e loca l quan t i f i ca ­t i on of oil r e m a i n i n g after spi l lage ( B r a g g et a l . , 1994), w h i c h essent ia l ly r equ i r e s a p p r o p r i a t e s a m ­p l ing a n d ana ly t i ca l s t ra teg ies . I n th i s sense , t h e a p p l i c a t i o n of G C - M S t e c h n i q u e s a n d b i o m a r k e r d e t e r m i n a t i o n s t h e r e f r o m ( H o s t e t t l e r a n d K v e n v -o lden , 1994; P o l l a r d et al . , 1999; P a g e et a l . , 2002 ; Pe t e r s a n d M o l d o w a n , 2004) is t h e m o s t p o p u l a r t o o l for e v a l u a t i n g c h a n g e s in t he chemica l c o m p o s i t i o n of oil de r iva t ives spil led in n a t u r a l e n v i r o n m e n t s .

A s m e n t i o n e d a b o v e , b i o d e g r a d a t i o n is o n e of t he m a i n p rocesses e n h a n c i n g n a t u r a l d e p l e t i o n of h y d r o c a r b o n s . I n th i s sense , t h e c o m p l e x c o m p o s i ­t i on of t h e Prestige fuel oil ( A l z a g a et a l . , 2004; Fa fe t et al . , 2004) impl ies t h a t on ly m i c r o b i a l m i x ­tu re s (genera l ly t e r m e d " c o n s o r t i a " ; H u r s t , 2002) a re c a p a b l e of p e r f o r m i n g effective b i o d e g r a d a t i o n in n a t u r a l e n v i r o n m e n t s ( B o u c h e z et a l . , 2000; K a n a l y et a l . , 2000; M i s h r a et al . , 2001) . I t is c lear t h a t i nd iv idua l m i c r o o r g a n i s m s c a n m e t a b o l i z e on ly a l imi ted r a n g e of h y d r o c a r b o n s u b s t r a t e s ( W a c k e t t a n d H e r s h b e r g e r , 2001) , so m i x e d p o p u l a t i o n s w i t h b r o a d e n z y m a t i c capac i t i e s a n d syne rg i s t i c / co -me ta -bo l i c r e l a t i o n s h i p s a r e r e q u i r e d t o a t t a c k n o t on ly a l i pha t i c a n d a r o m a t i c h y d r o c a r b o n s , b u t a l so N S O ( n i t r o g e n , sulfur, oxygen) c o m p o u n d s a n d a s p h a l t e n e f r ac t ions (Spe igh t , 2 0 0 1 ; V e n o s a a n d Z h u , 2003) .

B i o r e m e d i a t i o n s t ra teg ies inc lude n a t u r a l a t t e n u ­a t i on , as well as t h e acce l e ra t ion of b i o d e g r a d a t i o n by a d d i n g e x o g e n o u s m i c r o b i a l p o p u l a t i o n s ( b i o a u g m e n t a t i o n ) o r b y s t i m u l a t i n g i n d i g e n o u s p o p u l a t i o n s ( b i o s t i m u l a t i o n ) . B i o a u g m e n t a t i o n a p p r o a c h e s h a v e n o t b e e n successful a t Prestige sites ( M u r a d o et al . , 2004) a n d h a v e genera l ly b e e n ru l ed o u t for m a r i n e oil spill r e m e d i a t i o n (Lee et a l . ,

1997). O n t h e o t h e r h a n d , full scale b i o s t i m u l a t i o n in oil spills ( H e a d a n d S w a n n e l l , 1999; V e n o s a a n d Z h u , 2003) w a s first u s e d in A l a s k a w i t h t h e E x x o n V a l d e z spill , w h e r e t he a p p l i c a t i o n of o leoph i l i c a n d s low re lease fert i l izers ( B r a g g et al . , 1994; Z h u et al . , 2001) p l a y e d a n i m p o r t a n t ro le . Since t h e n , t h e b e n c h m a r k of m a r i n e oil spill b i o r e m e d i a t i o n is b a s e d o n s u p p l y i n g e x o g e n o u s n u t r i e n t s ( O h et al . , 2 0 0 1 ; M a k i et al . , 2003) . I n p a r t i c u l a r , o leoph i l i c fert i l izers h a v e b e e n r e p o r t e d as t h e o p t i m a l a l t e r n a ­t ive for b i o r e m e d i a t i n g r o c k y a r ea s in i n t e r t i da l zones . I n i p o l E A P 22 , des igned b y El f A q u i t a i n e after t he A m o c o C a d i z oil spill in F r a n c e in 1978, w a s successfully app l i ed in t h e r e m e d i a t i o n of s o m e E x x o n V a l d e z sites. Af te r t h e V a l d e z exper i ence ( P r i t c h a r d et al . , 1992), I n i p o l a n d s imi lar p r o d u c t s h a v e b e e n u s e d in o t h e r oil spills w i t h a c c e p t a b l e resu l t s ( S a n t a s et al . , 1999). H e n c e , o u r first o p t i o n w a s t o use a c o m m e r c i a l l y ava i l ab le o leoph i l i c fertil­izer - S200, whi le o t h e r poss ibi l i t ies , i n c l u d i n g t h e o n g o i n g n a t u r a l a t t e n u a t i o n s tud ies , w e r e be ing c o m p l e t e d .

2. Materials and methods

2.1. Site descriptions

T w o sites b e l o n g i n g t o o n e of t h e a r e a s m o s t severely affected by t h e Prestige fuel ( C o s t a d e la M u e r t e , L a Coru f i a , Spa in ) w e r e selected for th i s w o r k a n d for b i o r e m e d i a t i o n . A r o u n d the m i d d l e of N o v e m b e r 2002 , t he spil led fuel oil r e a c h e d " C o i d o d a C u r i o " a n d M o r e i r a B e a c h (F ig . 1), b o t h l o c a t e d o n t h e A t l a n t i c c o a s t of Ga l i c i a ( n o r t h w e s t ­e r n S p a i n ) , 80 k m f r o m L a Coru f i a . F u e l c o n t i n u e d t o a r r ive a t these sites in g r e a t q u a n t i t i e s f r o m N o v e m b e r 2 0 0 2 t o J a n u a r y 2 0 0 3 , cove r ing a cons id ­e r ab l e e x p a n s e of s e d i m e n t s a t t h e u p p e r t ida l l imit , co inc id ing w i t h m a x i m u m h i g h t ide . T h e r a t e of w a s t e a r r iva l d ec r ea s ed d r a m a t i c a l l y the rea f t e r a n d ini t ia l c l ean ing w o r k b e g a n (access w a y s w e r e i m p r o v e d , w a s t e c o n t a i n e r s w e r e ins ta l led , e tc . ) . By F e b r u a r y 2 0 0 3 , b o t h sites w e r e selected for r e sea r ch a n d b i o r e m e d i a t i o n p u r p o s e s b e c a u s e of the i r eco logica l a n d geo log ica l in te res t . I n M a r c h , t he p r e s e n t s t u d y c o m m e n c e d . A t t h a t p o i n t , t h e on ly c l e a n - u p p r o c e d u r e t h a t h a d b e e n i m p l e m e n t e d cons i s t ed of m a n u a l l y r e m o v i n g several t o n n e s of fuel cove r ing t h e s ed imen t s .

C o m m o n aspec t s of b o t h sites inc lude t h e he te r ­ogene i ty of r o u n d e d clas t ic s e d i m e n t g r a i n s , r a n g i n g f r o m a m i n i m u m of m e d i u m g r a i n size s a n d

( < 0 . 5 m m d i a m e t e r ) a t M o r e i r a B e a c h u p t o h u g e g ran i t i c b o u l d e r s (2 m d i a m e t e r a n d la rger ) a t t h e C u n o site. R e m a r k a b l y , these s e d i m e n t s a r e d i s t r ib ­u t e d a t b o t h sites o n t o p of t h i ck s t r a t a of n o n per ­m e a b l e o r g a n i c soil. T h u s , b o t h t h e h i g h viscosi ty of t he fuel a n d th i s geo log ica l s u b s t r a t e a t these sites i m p e d e h y d r o c a r b o n p e n e t r a t i o n - a n i m p o r t a n t difference w i t h respec t t o t h e E x x o n V a l d e z sites ( B r a g g et a l . , 1994). C o n s e q u e n t l y , o u r s t u d y focussed m a i n l y o n b r o a d sur face a r e a s c o v e r e d b y c o b b l e s ( b e t w e e n 64 a n d 256 m m d i a m e t e r ) a t t h e M o r e i r a site a n d ' sma l l ' b o u l d e r s ( b e t w e e n 256 a n d 800 m m d i a m e t e r a t t h e C u n o site). A s a final c o m m o n cha rac t e r i s t i c , smal l m e a n d e r i n g f reshwa­ter s t r e a m s flow o n t h e w e s t e r n s ide of t h e M o r e i r a b e a c h a n d o n t h e s o u t h e r n side of t he C u n o site.

T h e n o r t h e r n a r e a of M o r e i r a B e a c h is c o m p r i s e d of a t h i n layer of m e d i u m g r a i n s i l iceous s a n d o n a c o n s i d e r a b l e s lope t h a t r e m a i n s c o m p l e t e l y u n d e r w a t e r d u r i n g h i g h t ide . I n t h e s o u t h e r n , e a s t e r n a n d w e s t e r n a r ea s , t h e s a n d is c o v e r e d b y a r e g u l a r layer of p o r p h y d i c a n d g r an i t i c pebb l e s , c o b b l e s a n d b o u l d e r s de r ived f r o m geo log ica l s u b s t r a t e m e t e o r -i za t ion . T h e s e s h o r e r o c k s o c c u p y a n i r r egu l a r sur­face a r e a of ca . 2000 m 2 . F u r t h e r m o r e , t h e effects of t h e s t r o n g w i n d s t h a t a r e typ ica l of t h e a r e a gen­e r a t e d a smal l , e m e r g i n g d u n e sys tem a l o n g t h e t o p of t h e s lope w h e r e s o m e of t h e r o c k s a r e pa r t i a l l y b u r i e d in t h e s a n d .

T h e C o i d o d a C u n o site is l o c a t e d 5 k m n o r t h of M o r e i r a B e a c h a n d o p e n s o n t o t h e sea o n the wes t . Geo log ica l l y it is very s imi lar t o M o r e i r a , excep t t h a t t he g r a i n size of t h e s e d i m e n t s r a n g e s f r o m grave l pa r t i c les (2 m m m i n i m u m o n t h e n o r t h e r n side of t h e b e a c h ) u p t o l a rge r b o u l d e r s o n t h e s o u t h e r n side, w h i c h finally e n d s in a 2 0 - m ver t ica l g r a n i t e cliff. B o t h t h e cliff a n d t h e a r e a w h e r e t h e b o u l d e r s a re l o c a t e d lay o n i n t e r b e d d e d co luv ia l Q u a t e r n a r y s e d i m e n t s , m i x e d w i t h t h e a b o v e m e n ­t i o n e d o r g a n i c s u b s t r a t e a n d g ran i t i c p e b b l e s . S h o r e r o c k s (pebb les , c o b b l e s a n d b o u l d e r s ) cove r a n a p p r o x i m a t e surface a r e a m e a s u r i n g 400 m l o n g b y 20 m w i d e a t l ow t ide (8000 m 2 ) .

2.2. Fuel load quantification

I n t h e casu i s t ry de sc r ibed a b o v e , fuel quant i f ica­t i o n is p re fe rab ly exp res sed as fuel l o a d pe r sedi­m e n t sur face r a t h e r t h a n in t e r m s of fuel l o a d pe r s e d i m e n t m a s s ( this t ype of exp re s s ion s h o u l d b e rese rved on ly for s a n d y s ed imen t s ) . A d d i t i o n a l l y , t he se lect ion of p r o p e r C : N : P r a t i o s for b i o s t i m u l a ­

t i o n des ign r equ i r e s t h a t fuel l o a d be ca l cu l a t ed in o r d e r t o e s t i m a t e ava i l ab le o r g a n i c c a r b o n . T h u s , t w o p a r a m e t e r s w e r e n e e d e d t o quan t i fy t h e t o t a l a m o u n t of fuel r e m a i n i n g o n t h e s h o r e r o c k s ( r ega rd less of g r a in size): (i) t o t a l sur face a r e a cov­e red b y fuel a n d (ii) t o t a l fuel l o a d pe r r o c k sur face .

T o d e t e r m i n e t he t o t a l sur face a r e a c o v e r e d b y the fuel, special i m a g e ana lys i s t e c h n i q u e s w e r e app l i ed : t he affected sites w e r e d iv ided i n t o sub -a r ea s of 200 m 2 , t h e n t w o sphe res of k n o w n sur face a r e a w e r e r a n d o m l y i n c l u d e d in e a c h s u b - a r e a a n d r e p e a t e d d ig i ta l p h o t o g r a p h s of these s u b - a r e a s of e a c h site w e r e t a k e n t o c o m p l e t e t h e t o t a l sur face of t h e sites affected. A f t e r w a r d s , t he Split® (Spl i t E n g i n e e r i n g 2 0 0 1 , A r i z o n a , U S A ) so f tware w a s u s e d for d igi ta l ana lys i s of t h e i m a g e s t a k e n . T h i s p r o g r a m m e ca lcu la tes g r a in size d i s t r i b u t i o n cu rves a s s u m i n g t h a t e a c h r o c k c o u l d b e ass imi la ted i n t o a n el l ipsoid h a v i n g t h e s a m e a r e a a n d f ac to r ing in t he k n o w n a r e a of t h e sphe res t h a t a p p e a r in e a c h i m a g e . Successive c a l c u l a t i o n s of all t h e s u b - a r e a s of t h e b o u l d e r - c o b b l e - p e b b l e fields e s t i m a t e a n a p p r o x i m a t i o n t o t he t o t a l sur face a r e a of fuel-cov­e red r o c k s .

A s ta t i s t ica l m e a s u r e m e n t of t h e fuel layer th ick­nesses w a s t h e n n e e d e d t o finally d e t e r m i n e fuel l o a d pe r u n i t of r o c k sur face . T h i s s e c o n d p a r a m e t e r w a s ca l cu l a t ed b y a v e r a g i n g t h e we igh t s of fue l - sampl ing i n c r e m e n t s t a k e n b y s c r a p i n g t h e r o c k s : t w e n t y dif­ferent o p e r a t o r s s c r aped d u p l i c a t e s of 4 c m 2 (2 c m side squa res ) of fuel f r o m t en r o c k s r a n d o m l y dis ­t r i b u t e d a t t h e C u n o site; t h e s a m e o p e r a t i o n w a s p e r f o r m e d a t t h e M o r e i r a site. T h e w e i g h t s of t h e i n c r e m e n t s w e r e a v e r a g e d ; th i s des ign t h e r e b y c o n ­s ide rab ly m i n i m i z e d s t a n d a r d d e v i a t i o n . S imi la r p r o c e d u r e s w e r e u s e d w h e n neces sa ry t o specify resu l t s in ce r t a in site s u b - a r e a s .

2.3. Sampling methodology and strategies

S y s t e m a t i c s a m p l i n g of t he s u b - a r e a s w a s c o n ­d u c t e d fo l lowing t he gene ra l p r o c e d u r e de sc r ibed a b o v e for fue l -covered r o c k s , cons i s t ing of s c r a p i n g d u p l i c a t e c o m p o s i t e s amp le s . E a c h c o m p o s i t e s a m ­ple c o m p r i s e d t en i n c r e m e n t s of 4 c m 2 of fuel geo­me t r i ca l ly d i s t r i b u t e d a t s a m p l i n g s t a t i o n s , c o r r e s p o n d i n g t o t en r o c k s (pebb le s , c o b b l e s , o r b o u l d e r s d e p e n d i n g o n t h e s u b - a r e a s a m p l e d ) . T h e i n c r e m e n t s w e r e t a k e n by s c r a p i n g t h e fuel c o v e r i n g 4 c m 2 of r o c k us ing a p p r o p r i a t e m e t a l t o o l s , avo id ­ing t h e co l lec t ion of r o c k f r a g m e n t s , a lgae , o r a n y o t h e r i m p u r i t y as far as poss ib le . F o r oily s a n d y

s e d i m e n t s a t t h e M o r e i r a site a n d oily grave l sedi­m e n t s a t t h e C u n o site respect ively , t h e s t r a t egy fol­l o w e d w a s t h e s a m e as de sc r ibed for t h e r o c k s (except for s a m p l e we igh t ) ; t he re fo re , a p p r o x i m a t e l y 100 g for s a n d y s e d i m e n t s w e r e w e i g h e d whi le 15 g were to t a l ly t a k e n f r o m fuel cove r ing sho re r o c k s . T h e s a m p l e s w e r e t h e n p l a c e d in c o n t a i n e r s w h i c h were sea led a n d s t o r e d in t h e d a r k a t 4 °C . W h e n s a m p l e s w e r e n e e d e d for m i c r o b i o l o g i c a l d e t e r m i n a ­t i ons ; t h e y w e r e t a k e n a n d s t o r e d in sterile c o n d i ­t i ons . S o m e m o d i f i c a t i o n s t o these gene ra l p r o t o c o l s w e r e a p p l i e d t o specific s a m p l i n g s t a t i o n s a n d a r e desc r ibed be low .

2.4. Hydrocarbon analysis

T h e ana lys i s of t h e fuel s a m p l e s s c r a p e d f r o m t h e r o c k s w a s p e r f o r m e d as fo l lows: T h e h y d r o c a r b o n s were e x t r a c t e d f r o m a 1 g s a m p l e w i t h C H 2 C 1 2 in a 50 m l Soxh le t a p p a r a t u s for 24 h ( E P A M e t h o d 3540 C ) . Af te r so lven t e v a p o r a t i o n a n d r e d i l u t i o n in d i c h l o r o m e t h a n e , 2 m l ex t r ac t s w e r e puri f ied u s ing l iqu id c h r o m a t o g r a p h y w i t h « -hexane as sole so lvent ( E P A M e t h o d 3630 C ) . Pur i f ied e x t r a c t s ( c o n t a i n i n g on ly a l i pha t i c a n d a r o m a t i c f rac t ions) were injected i n t o a G C sys t em ( H P 6890 Series c h r o m a t o g r a p h ) e q u i p p e d w i t h a cap i l l a ry c o l u m n ( A T 5 Al l t ech ; 25 m x 0.25 m m i .d.) ; t h e oven t e m ­p e r a t u r e w a s ra i sed f r o m 60 ° C t o 300 ° C a t 6 ° C / m i n . T h e in jec tor t e m p e r a t u r e w a s 275 °C . T h i s e q u i p m e n t w a s a t t a c h e d t o a M S D H P 5973 Series m a s s d e t e c t o r , w h i c h a l l owed p e a k a r e a d e t e r m i n a ­t i ons in selective i o n m o n i t o r i n g ( S I M ) m o d e us ing the Wi ley 275 d a t a b a s e ( E P A M e t h o d 8270 C ) . A n a l y s i s of s a n d y s e d i m e n t s differed on ly w i t h r e g a r d t o t he w e i g h t of t he ini t ia l s u b - s a m p l e (20 g) e x t r a c t e d in t he Soxh l e t a p p a r a t u s .

Different r a t i o s w e r e c a l c u l a t e d f r o m t h e d a t a in G C - M S c h r o m a t o g r a m s to m e a s u r e w e a t h e r i n g a n d b i o d e g r a d a t i o n . T h e y w e r e ca l cu l a t ed after careful ly s t u d y i n g t h e s a m p l e s t a k e n d u r i n g t h e first m o n t h s of fieldwork a n d a re de sc r ibed be low .

2.5. Microbiological methods

M i c r o b i a l c o u n t s w e r e p e r f o r m e d as fo l lows: a r ep re sen t a t i ve a l i q u o t (1 g) of e a c h col lec ted fuel s a m p l e w a s a d d e d t o 10 m l of a steri le 0 . 1 % N a 2 H P 0 4 - 1 2 H 2 0 s o l u t i o n a n d m i x e d (vo r t ex stir­rer) v igo rous ly for 10 m i n a c c o r d i n g t o s t a n d a r d p r o c e d u r e s for soil s a m p l e s ( G a l l e g o e t a l . , 2001) . A n a l i q u o t (1 ml ) of t he s u p e r n a t a n t s u s p e n s i o n

(a lso u s e d as i n o c u l u m for selective e n r i c h m e n t ; see be low) w a s t a k e n a n d 0.1 m l of successive di lu­t i o n s of t h e c u l t u r e w e r e s p r e a d in t r ip l i ca te T S B ( t ryp t i c soy b r o t h , M e r c k ) a g a r p l a t e s , supp le ­m e n t e d w i t h 2 % N a C l a n d 0 . 1 % yeas t e x t r a c t a n d cu l t i va t ed a t 30 ° C for 48 h in a e r o b i c c o n d i t i o n s ( n o t e t h a t th i s m e d i u m a n d o t h e r s desc r ibed b e l o w m a y c a u s e in s o m e cases a r e d u c e d cell c o u n t d u e t o u n u s u a l g r o w t h c o n d i t i o n s ) . F o r fungi g r o w t h , d u p l i c a t e s w e r e s p r e a d in p o t a t o a g a r m e d i u m ( O x o i d ) s u p p l e m e n t e d w i t h 2%> N a C l . T h e p r e d o m ­i n a n t s t r a ins w e r e a lso i so l a t ed in T S B m e d i u m a n d t es ted in diesel a g a r syn the t i c m e d i u m ( D S ) : 0.13%> N H 4 N 0 3 ; 0 . 0 5 % M g S 0 4 H 2 0 ; 0 . 0 2 % C a C l 2 - 2 H 2 0 ; 0 . 5 % K H 2 P 0 4 ; 0 . 5 % K 2 H P 0 4 a n d 2 % filter-steril­ized diesel fuel as sole c a r b o n sou rce .

Selective e n r i c h m e n t t e c h n i q u e s w e r e c o n d u c t e d in l iqu id syn the t i c m e d i u m h a v i n g t h e s a m e c o m p o ­s i t ion as D S sol id m e d i u m , excep t t h a t t h e diesel w a s s u b s t i t u t e d for 1%> Prestige fuel as sole c a r b o n sou rce ; 2 m l of t he i n o c u l a o b t a i n e d as a b o v e w e r e a d d e d t o 100 m l of m e d i u m a n d i n c u b a t e d in a r o t a r y s h a k e r a t 250 r p m a n d 30 ° C for 5 d. T h e n , 0.1 m l of t he s u s p e n s i o n w a s u s e d for p l a t e c o u n t i n g in T S B - a g a r m e d i u m a n d 2 m l w e r e u s e d as a s e c o n d i n o c u l u m for a n o t h e r flask c o n t a i n i n g t h e s a m e m e d i u m . T h e m o s t a b u n d a n t s t r a ins after t h r e e cul­t i va t i on s teps w e r e i so la ted a n d p r e se rved b y freez­ing a t —70 ° C in 1.7% t r e h a l o s e a n d 25%> glycerol m e d i u m .

Bas ic m o r p h o l o g i c a l d a t a r e g a r d i n g b a c t e r i a , fungi a n d c y a n o b a c t e r i a a n d the i r b e h a v i o u r in n a t ­u r a l fue l - con ta in ing m e d i a w e r e o b t a i n e d b y m e a n s of p h a s e c o n t r a s t m i c r o s c o p y ( N i k o n Ecl ipse E-200) . Bac te r i a l v iabi l i ty w a s a n a l y z e d b y s t a in ing the cells w i t h p r o p i d i u m iod ide (PI ) a n d S Y T O 9 g reen f luorescent nuc le ic ac id s t a in ( L I V E / D E A D B a c - L i g h t Bac te r i a l Viab i l i ty K i t , M o l e c u l a r P r o b e s ) . S a m p l e s w e r e e x a m i n e d u n d e r a Le ica T C S - S P 2 - A O B S confoca l l a s e r - s cann ing m i c r o ­scope ( C L S M ) a t t h e a p p r o p r i a t e w a v e l e n g t h , a n d the i m a g e s w e r e m i x e d us ing t h e Le ica C o n f o c a l So f tware .

2.6. Biostimulation experiments with oleophilic fertilizer S-200

T h e o leoph i l i c fertil iser S200 ( I E P E u r o p e ) is l is ted in t h e N C P ( N a t i o n a l Oil a n d H a z a r d o u s S u b ­s t ances P o l l u t i o n C o n t i n g e n c y P l a n , U S A ) as a b i o ­r e m e d i a t i o n agen t . S200 (D iez e t al . , 2005) is a m i c r o e m u l s i o n of a s a t u r a t e d s o l u t i o n of u r e a ( N

source) in oleic ac id c o n t a i n i n g p h o s p h a t e es ters ( P source ) . T o tes t i ts effectiveness for b i o r e m e d i a t i o n p u r p o s e s , t h r e e s e p a r a t e 200 m 2 s u b - a r e a s of M o r e ­i r a B e a c h w e r e selected a n d subd iv ided ; ha l f received S-200 fertil izer a n d ha l f r e m a i n e d as u n t r e a t e d c o n t r o l s . S-200 w a s a p p l i e d m o n t h l y w i t h a m e c h a n i c a l sp raye r t h r e e t i m e s d u r i n g t h e s u m m e r of 2 0 0 3 , in t o t a l d o s a g e s a c c o r d i n g to t h e des i r ab l e C : N : P r a t i o of 100:10:1 ( H e a d a n d Swanne l l , 1999; Z h u et al . , 2001) a n d b a s e d o n t h e p rev ious ly ca lcu­l a t ed fuel l o a d affecting t h e shore l ines .

3. Results and discussion

3.1. Fuel characterization and definition of depletion indexes

T h e o r ig ina l fuel oil h a s b e e n r e p e a t e d l y a n a l y s e d b y different a u t h o r s ; for i n s t ance , S A R A ( s a t u r a t e ,

a r o m a t i c , res ins a n d a s p h a l t e n e ) f r a c t i o n a t i o n revea led c o n c e n t r a t i o n s of a p p r o x i m a t e l y 2 5 % sa tu ­r a t e d h y d r o c a r b o n s , 35%> a r o m a t i c s , 20%> res ins a n d 20%> a s p h a l t h e n e s ( A l z a g a et al . , 2004; F a f e t et al . , 2004) . T h i s a p p r o a c h revea led t h a t w e w e r e d e a l i n g w i t h a h e a v y r e s idue f r o m a t m o s p h e r i c d i s t i l l a t ion , u sua l ly classified as fuel n °6 . M o r e o v e r , o u r ini t ia l G C - M S s tud ies of fuel t a k e n d i rec t ly f r o m t h e sh ip a t t h e b e g i n n i n g of t h e spill s h o w e d t h a t a l k y l a t e d P A H s a n d heav ie r l inear a n d b r a n c h e d a l k a n e s ( C i 2 - C 4 0 ) w e r e t h e m o s t a b u n d a n t famil ies in t h e s a t u r a t e a n d a r o m a t i c f rac t ions (as p rev ious ly r e p o r t e d b y A l z a g a et al . (2004) , B e n o i t a n d H a e s -eler (2004)) .

F o r t he p r e s e n t s tudy , i n t e r n a l s t a n d a r d s such as i s o p r e n o i d s ( p r i s t a n e a n d p h y t a n e ) a n d h o p a n e s n e e d e d to b e d e t e r m i n e d . P r i s t a n e a n d p h y t a n e a r e useful in t he ini t ia l s tages of t he d e g r a d a t i o n p r o ­cesses t o m o n i t o r « - a l k a n e dep l e t i on ; h o w e v e r , as

17a, 21P(H) hopane

n-Ci,

17a, 21P(H) -30-Norhopane

Tm

n-C,

apc 3i

apc 3 :

aPC3!

01PC34

10.00 15.00 20 .00 25 .00 30 .00 35 .00 40 .00 45 .00

00 36 .00 38 .00 4 0 . 0 0 42 .00 44 .00 46 .00 48 .00 50 .00

Fig. 2. GC-MS chromatograms of initial it Prestige fuel oil: (a) total ion chromatogram (TIC) mode, (b) selective ion mode (SIM, m/z 57) showing «-alkanes, (c) m/z 191 SIM showing hopane fingerprinting. (Ts: 18a(H)-22,29,30-trisnorhopane; Tm:17oi(H)-22,29,30-trisnorhopane; 31afS, 32 afS, 33 afS and 34 afS are pairs of R and S isomers of 17a(H), 21fS(H), 22-homohopane, 17a(H), 21fS(H), 22-bishomohopane, 17a(H), 21fS(H), 22-trishomohopane and 17a(H), 21fS(H), 22-tetrakishomohopane respectively).

r e p o r t e d p rev ious ly (Sasak i et al . , 1998; W a n g a n d F i n g a s , 2003) a n d a lso d e m o n s t r a t e d b e l o w , t h e y a re of ten d e g r a d e d m o r e qu ick ly t h a n is genera l ly t h o u g h t . O n the o t h e r h a n d , n o r m a l i z a t i o n t o h o p a n e h a s b e e n r e p o r t e d as t h e bes t m e t h o d for quan t i fy ing h y d r o c a r b o n d e p l e t i o n a n d the re fo re b i o d e g r a d a t i o n , specifically t h e use of 1 7 a ( H ) , 21 p ( H ) - h o p a n e as a n i n t e r n a l c o n s e r v a t i v e s t a n d a r d ( B r a g g et al . , 1994; P r ince et al . , 1994; P o l l a r d e t a l . , 1999; Pe t e r s a n d M o l d o w a n , 2004) . F u r t h e r m o r e , 1 7 a ( H ) , 2 1 p ( H ) - h o p a n e w a s c lear ly t he m o s t a b u n ­d a n t t e r p a n e in t h e Prestige fuel oil, fo l lowed b y 17a (H) 2 1 p ( H ) - 3 0 - n o r h o p a n e (F ig . 2) , a n d its ex t re ­me ly l ow solubi l i ty , b i o d e g r a d a b i l i t y a n d vola t i l i ty g u a r a n t e e t h a t it w o u l d pers i s t in t he r e s idua l fuel o n shore l ine s e d i m e n t s . C o n s e q u e n t l y , it w a s selected as a n i n d i c a t o r of t he p r o g r e s s m a d e w i t h b i o r e m e d i a t i o n t r e a t m e n t r e su l t ing f r o m t h e E x x o n V a l d e z oil spill in A l a s k a (P r ince et al . , 1994). I t h a s since b e e n u s e d t o quan t i fy t h e ex t en t of b i o d e g r a ­d a t i o n in m a n y o t h e r s tud ies . H o p a n e d e g r a d a t i o n h a s b e e n de sc r ibed (Bos t et al . , 2001) t o o c c u r in h igh ly specific l a b o r a t o r y c o n d i t i o n s a n d anc i en t oil r e se rvo i r s , a l t h o u g h never in oil spills s imi lar t o t he Prestige o n e . I n a n y even t , even if h o p a n e w e r e sl ightly d e g r a d e d d u r i n g b i o t r a n s f o r m a t i o n , t h e effects w o u l d on ly sl ightly u n d e r e s t i m a t e t h e a c t u a l oil dep l e t i on .

In i t ia l ly , c o m p a r i s o n of t h e r a t i o s of several ind i ­v idua l c o m p o u n d s o r g r o u p s of c o m p o u n d s w a s c h o s e n t o define d e p l e t i o n r a t i o s . Se lec t ion c r i t e r i a cons i s t ed of a b u n d a n c e in Prestige fuel, r e a d y loca­t i on a n d iden t i f ica t ion in S I M c h r o m a t o g r a m s a n d

rep resen t iv i ty of s o m e of t h e m o s t i m p o r t a n t fami ­lies in fuel oil (Spe igh t , 2001) . T h e list i nc luded lin­ea r a l i pha t i c h y d r o c a r b o n s of l i g h t - m e d i u m m o l e c u l a r w e i g h t ( h e p t a d e c a n e a n d o c t a d e c a n e ) , w a x e s r a n g i n g f r o m t r i a c o n t a n e t o t r i t r i a c o n t a n e , i s o p r e n o i d s (p r i s t ane a n d p h y t a n e ) , t h r e e r ing a lkyl P A H s ( m e t h y l a n d d i m e t h y l a n t h r a c e n e s a n d p h e -n a n t h r e n e s - C l - A P h a n d C 2 - A P h ) , p y r e n e ( four r ing P A H ) , a lkyl de r iva t ives of t h e m o s t a b u n d a n t sulfur c o m p o u n d s ( d i m e t h y l d i b e n z o t h i o p h e n e s -C 2 - D B T ) a n d finally, 1 7 a ( H ) , 2 1 p ( H ) h o p a n e ; all h a v e b e e n wide ly u s e d for e s t ab l i sh ing d e p l e t i o n r a t i o s a n d for o t h e r s imi lar p u r p o s e s (Pe te r s a n d M o l d o w a n , 2004) .

I t is c lear t h a t a n i m p o r t a n t spa t i a l va r iab i l i ty in t he ana lyses w o u l d b e expec t ed ( P a g e et al . , 2002) . A t t h e Prestige si tes, th i s is a c c o u n t e d for by sev­era l f ac to r s i n c l u d i n g t ida l r e a c h , different da i ly h o u r s of sun ( p r o m o t i n g o r dep le t i ng vo la t i l i s a t ion of l ight c o m p o u n d s a n d / o r p h o t o c h e m i c a l ox ida ­t i o n of a r o m a t i c s ) , g r a in size, p r e sence o r a b s e n c e of s a n d y o r g rave l s e d i m e n t s c o a t i n g t he p o l l u t e d r o c k s , exis tence of a r ea s w i t h p re fe ren t ia l c i rcula­t i o n of r a i n w a t e r s , e tc . T h e r e f o r e , for c ross site r a t i o var iab i l i ty , a s a m p l i n g p r o c e d u r e w a s per ­f o r m e d b y select ing several 200 m 2 s u b - a r e a s a t t he Cur io si te. T h r e e r ep l i ca te s a m p l e s w e r e t a k e n f r o m five of these s u b - a r e a s , c o m p r i s i n g 10 g e o ­me t r i ca l ly d i s t r i b u t e d fuel s u b - s a m p l e s , s c r a p e d f r o m 4 c m 2 sur faces as e x p l a i n e d a b o v e . T h u s , a t o t a l of 15 s a m p l e s w a s t a k e n a n d ana ly sed us ing G C - M S ( T a b l e 1). T h e r a t i o s of different c o m ­p o u n d s in t h e fuel oil w e r e c a l c u l a t e d b y a v e r a g i n g

Table 1 Depletion ratios obtained by averaging results of 15 samples taken simultaneously at Cuno site in July 2003

Ratio Mean value ± std. error Relative std. error (%)

Isoprenoid normalization C17/pristane 0.41 ± 0.24 59.06 C18/phytane 0.45 ± 0.20 46.64 C17 + C18/pristane + phytane 0.43 ± 0.22 50.07 C30 + C31 + C32 + C33/Pristane + phytane 2.28 ± 1.12 49.17

Hopane normalization C17 + C18/17oi2ip hopane 1.52 ±0.48 31.58 C30 + C31 + C32 + C33/17a2ip hopane 8.12 ± 1.41 17.37 Pristane + phytane /17a21fS hopane 3.75 ±0.63 16.86 ]T(C r APh + C2-APh)/17oi2ip hopane 15.36 ±2.91 18.93 £C 2-DBT/17oi2ip hopane 3.71 ±0.73 19.57 Pyrene/17a2ip hopane 1.13 ±0.20 17.81

Others Pristane/phytane 0.78 ± 0.07 9.05 £ C r A P h / £ C 2 - A P h 0.82 ±0.07 8.68 ]TC 2-APh/]TC 2-DBT 2.35 ±0.42 18.06

Ci-APh: methyl anthracenes and phenanthrenes; C2-APh: dimethyl anthracenes and phenanthrenes; C 2-DBT: dimethyldi-benzothiophenes.

t he G C - M S d a t a after n o r m a l i z a t i o n w i t h t h e i n t e rna l s t a n d a r d s . R e s u l t s s h o w e d signif icant he t ­e rogene i ty , i nd i ca t i ng t h a t different w e a t h e r i n g p rocesses w e r e t a k i n g p lace in a n a p p a r e n t l y h o m o g e n e o u s s cena r io . H o w e v e r , loca l v a r i a t i o n s in t h e s u b - a r e a s w e r e c o n s i d e r a b l y smal le r t h a n t h o s e o b t a i n e d for t he overa l l site. T h e r e b y , spe­cific s u b - a r e a s w e r e selected for m o n i t o r i n g t h e c h a n g e s in fuel c o m p o s i t i o n over t i m e . N o t e t h a t , as r e p o r t e d in T a b l e 1, t h e r a t i o s w i t h g r e a t e r degrees of u n c e r t a i n t y a r e t h o s e t h a t i nc lude l ight a n d easy d e g r a d a b l e h y d r o c a r b o n s such as « - C i 7

a n d K-Cig a l k a n e s . I n a d d i t i o n , it seems t h a t t h e va lues o b t a i n e d us ing p r i s t a n e a n d p h y t a n e as h y p o t h e t i c a l n o n - d e g r a d a b l e s t a n d a r d s f luc tua ted m u c h m o r e ( re la t ive s t a n d a r d e r r o r > 45%) t h a n t h o s e o b t a i n e d w i t h 1 7 a ( H ) , 2 1 p ( H ) h o p a n e (re la­

t ive s t a n d a r d e r r o r 16 .86% t o 50.07%>); h e n c e , t h e f o r m e r w e r e ru l ed o u t a n d t h e l a t t e r selected for fuel e v o l u t i o n ana lys i s . I t w o u l d b e r e a s o n a b l e t o cons ide r these ' h o p a n e r a t i o s ' t o b e s y n o n y m o u s w i t h b i o t r a n s f o r m a t i o n r a t i o s , a s s u m i n g t h a t , a t t he b e g i n n i n g of o u r s t u d y ( M a r c h 2003) , phys ica l w e a t h e r i n g ( v a p o r i z a t i o n a n d d i s so lu t ion ) of t h e Prestige fuel oil w a s a l m o s t negl igible in m o s t sites. T h e s a m e a s s u m p t i o n w a s d i scussed in b i o r e m e d i a -t i o n s tud ies for N o r t h S lope c r u d e oil a t t h e E x x o n V a l d e z sites ( B r a g g et al . , 1994). I n a n y case , t h e c o m p l e x p rocesses imp l i ed in n a t u r a l a t t e n u a t i o n , t he difficulties in d e t e c t i o n of p a t h w a y i n t e r m e d i ­a tes a n d t h e non- feas ib i l i ty of p e r f o r m i n g a b i o t i c e x p e r i m e n t s a t field si tes, sugges t t o u s t h e use of t he t e r m " d e p l e t i o n r a t i o s " i n s t e a d of " b i o t r a n s ­f o r m a t i o n r a t i o s " .

17+C18/hopane

+C31 +C32+C33/hopane

- Ph /hopane

May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04 Jul-04 Sep-04 Nov-04 Jan-05 Mar-05

Z (C1-APh +C2-APh)/hopane

Z C2-DBT/hopane

Pyrene /hopane

May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04 Jul-04 Sep-04 Nov-04 Jan-05 Mar-05

Fig. 3. Temporal fate of depletion indexes calculated for samples taken at Cuno site over a 2 year period. Errors are within % values in Table 1. (Pr: pristane, Ph: phytane; Cj-APh: methyl anthracenes and phenanthrenes; C2-APh: dimethyl anthracenes and phenanthrenes; C2-DBT: dimethyldibenzothiophenes; hopane: 17a(H), 21fS(H), hopane).

Fina l ly , a n d t o faci l i tate u n d e r s t a n d i n g , dep le ­t i on r a t i o s w e r e mod i f i ed t o p r o v i d e d e p l e t i o n indexes ( P o l l a r d et al . , 1999; W h i t t a k e r et a l . , 1999) as a p e r c e n t a g e of d e p l e t i o n for t h e different famil ies of c o m p o u n d s . F o r e x a m p l e , t h e t e r m 'pyr -ene d e p l e t i o n i n d e x ' is expressed in t e r m s of c o n c e n ­t r a t i o n of p y r e n e a n d 1 7 a ( H ) , 2 1 p ( H ) h o p a n e as :

P y r e n e d e p l e t i o n i n d e x ( % )

/ ( p y r e n e / 1 7 a 2 1 p h o p a n e ) , a m r f p \ = 100 - — p • 100

V ( p y r e n e / 1 7 a 2 1 p h o p a n e ) r e f e r e n c e J

3.2. Evolution of fuel residues

F u e l r e s idue fa te a t t he Cur io site w a s invest i ­g a t e d over a t w o year p e r i o d b y m e a n s of p e r i o d i c ana lys i s of t r ip l ica te s a m p l e s t a k e n a t t h r e e 200 m 2

a rea s of p o l l u t e d s h o r e r o c k s ( the res t of t h e site w a s u s e d for several o n - g o i n g b i o r e m e d i a t i o n exper ­imen t s ) . D e p l e t i o n indexes w e r e a v e r a g e d t o o b t a i n r ep re sen t a t i ve va lues a p p l i c a b l e t o t he w h o l e si te. T h e resu l t s a r e s u m m a r i z e d in F ig . 3 , w h e r e s u b ­s t an t i a l d e g r a d a t i o n of all t h e h y d r o c a r b o n famil ies

Table 2 Depletion ratios for Cuno site from triplicate samples taken at 3200 m 2 areas of polluted shore rocks during a 2-year period

Ratio Pr/Ph ]TCl-APh/X;C2-APh ]TC2-APh/X;C2-DBT Mar-03 0.82 0.84 2.23 May-03 0.78 0.81 2.12 Jul-03 0.70 0.82 2.35 Sep-03 0.63 0.62 2.25 Nov-03 0.67 0.67 2.21 May-04 0.59 0.70 2.48 Jul-04 0.44 0.58 2.28 Sep-04 0.43 0.58 2.60 Feb-05 0.37 0.61 2.37

s tud i ed d u r i n g t h e first s u m m e r after t h e spi l lage is c lear ly seen. T h e d e p l e t i o n w a s p a r t i c u l a r l y ev iden t n o t on ly in t h e case of t he l ighter « - a l k a n e s , as expec t ed in n a t u r a l e n v i r o n m e n t s ( G h a z a l i et al . , 2004) , b u t a l so in t h e a r o m a t i c f rac t ion . H o w e v e r , t he p r o c e s s s lowed d o w n a n d m o r e t h a n o n e yea r after t h e e n d of t h e s u m m e r of 2 0 0 3 , t h e levels of t he s tud i ed c o m p o u n d s in t h e a r o m a t i c f r ac t ion r e m a i n e d s tab le , w h e r e a s a l ipha t i c s w e r e d e g r a d e d s o m e w h a t fur ther . I t is i n t e re s t ing t o p o i n t o u t t h a t t he r a t e of d e g r a d a t i o n of heav ie r « - a lkanes is s im­ilar t o o r even less t h a n t h a t for i s o p r e n o i d s . T h i s resu l t e m p h a s i z e s t h e differences b e t w e e n in s i tu b i o ­d e g r a d a t i o n a n d c o n t r o l l e d ex s i tu t r e a t m e n t a n d l a b o r a t o r y s tudies , in w h i c h a « - a l k a n e ( i r respec t ive of c h a i n l eng th) is genera l ly a s s u m e d to b e m o r e b i o d e g r a d a b l e t h a n a n i s o p r e n o i d . O n the o t h e r h a n d , s o m e of t h e f luc tua t ions in indices for a r o m a t -ics a n d b r a n c h e d a l k a n e s (seen in F ig . 3) , m a y b e a t t r i b u t a b l e t o t he s t a n d a r d e r r o r s s h o w n in T a b l e 1, a l t h o u g h in te r fe rence f r o m res in a n d a s p h a l t e n e f rac t ions m i g h t a l so b e a poss ib le e x p l a n a t i o n . I n th i s sense , t h e p r e s u m e d a p p e a r a n c e of s a t u r a t e s a n d a r o m a t i c s as a resu l t of a s p h a l t e n e p h o t o b i o o x -i d a t i o n p rocesses ( P i n e d a - F l o r e s et al . , 2004) a n d the poss ib le g r o w t h of t h e res in f r ac t ion v ia a r o ­m a t i c p h o t o o x i d a t i o n ( Jezeque l et al . , 2003) s h o u l d b e cons ide red .

F u r t h e r m o r e , t he i n f o r m a t i o n in T a b l e 2 c o m ­ple tes t h e d a t a a n d r e p o r t s fas ter d e g r a d a t i o n for p r i s t a n e vs . p h y t a n e a n d for C r P A H s vs . C 2 - P A H s , w h e r e a s C 2 - d i b e n z o t h i o p h e n e s a n d C 2 - P A H s h a v e s imi lar d e g r a d a t i o n r a t e s ( N . B . p r i s t a n e / p h y t a n e a n d J ] C i - A P h / J ] C 2 - A P h r a t i o s d i m i n i s h over t i m e , w h e r e a s J ] C 2 - A P h / J ] C 2 - D B T s h o w s on ly sl ight f luc tua t ion) . Be t h a t as it m a y a n d c o n s i d e r i n g t h e overa l l d a t a (F ig . 3 a n d T a b l e 2) , a c lassif icat ion of d e p l e t i o n suscept ib i l i ty for c o m p o u n d s in t h e Prestige fuel oil a t n a t u r a l si tes c a n b e p r o p o s e d :

Table 3 Depletion ratios for a 2 cm deep fuel core taken at a sampling point in the most highly polluted zone (granite cliff) at Cuno site (Sept., 2003)

Fuel compound External section Intermediate section Internal section

C17 + C18/17oi2ip hopane 3.32 5.67 6.30 C30 + C31 + C32 + C33/17a2ip hopane 5.08 8.58 9.89 pristane + phytane /17a2ip hopane 3.05 4.64 4.67 ]TCi-APh + c 2-APh /17a2ip hopane 19.45 31.32 32.82 £C 2-DBT/17oi2ip hopane 4.51 6.92 7.65 pyrene/17 oi2 ip hopane 1.28 1.78 1.75 pristane/phytane 0.77 0.86 0.92 EQ-APh/ECz-APh 0.86 0.95 0.99

l i g h t - m e d i u m « - a l k a n e s > b r a n c h e d a l k a n e s (pr i s ­t a n e > p h y t a n e ) > h e a v y l inear a l k a n e s (wax­es) > C i - t h r e e r i n g P A H s > C 2 - t h r e e r ing P A H s ~ C 2 - d i b e n z o t h i o p h e n e s > fou r r ing P A H s .

Al l of th i s sugges t s t h a t t he m i c r o o r g a n i s m s a t t he p o l l u t e d sites c o u l d po t en t i a l l y d e g r a d e a l i p h a t -ics extensively whi le a t t he s a m e t i m e s lowly d e g r a d ­ing t h e a r o m a t i c f rac t ion . T h e s e resu l t s a r e s o m e w h a t cons i s t en t w i t h t h o s e o b t a i n e d b y M e d -ina-Bel lver e t al . (2005) w h o d e m o n s t r a t e d h y d r o ­c a r b o n d e p l e t i o n a t Prestige sites b y m e a n s of i s o t o p i c f r a c t i o n a t i o n . T h e y a lso i so l a t ed several s t r a ins c a p a b l e of d e g r a d i n g a l ipha t i c s a n d s o m e a r o m a t i c s . N e v e r t h e l e s s , t he hos t i l e e n v i r o n m e n t of s h o r e r o c k s e x p o s e d t o s t r o n g w i n d s , sea t ides , l ow n u t r i e n t levels a n d o t h e r h a r s h c o n d i t i o n s m a y conce ivab ly h i n d e r o r even i m p e d e the b i o d e g r a d a -t i o n of t a r g e t e d c o m p o u n d s . T h u s , t he ex is tence of i m p o r t a n t a m o u n t s of still fresh, l ight h y d r o c a r ­

b o n s , t o g e t h e r w i t h t h e h i g h t e m p e r a t u r e s t o r e d u c e fuel v iscosi ty , f a v o u r i n g m i c r o b i a l g r o w t h , facili­t a t e d b i o d e g r a d a t i o n on ly d u r i n g t h e first s u m m e r . A d d i t i o n a l l y , a t m a n y l o c a t i o n s , t h e c o n s i d e r a b l e t h i cknes s of fuel c o a t i n g d i d n o t a l l ow sufficient p e n e t r a t i o n of o x y g e n , n u t r i e n t s , w a t e r a n d m i c r o ­o r g a n i s m s ; c o n s e q u e n t l y on ly t h e superf icial l ayers w e r e effectively w e a t h e r e d . T h i s w a s verified b y s a m ­p l ing a n d a n a l y s i n g t h e ex t e rna l , i n t e r m e d i a t e a n d i n t e r n a l sec t ions of a 2 c m d e e p fuel co r e e x t r a c t e d f r o m t h e ver t ica l g r a n i t e cliff a t t h e C u n o site; t h e resu l t s c lear ly s h o w e d a g r a d i e n t of dec rea s ing effect of d e g r a d a t i o n ( T a b l e 3) .

3.3. Natural attenuation vs. bioremediation with S200

U s i n g t h e s a m e a p p r o a c h as for t h e C u n o site, several pa rce l s w e r e selected a t M o r e i r a B e a c h to m o n i t o r n a t u r a l a t t e n u a t i o n of pe r iod ica l ly s a m p l e d

200 400 600 800 1000 1200 1400 1600 1800 2000

Grain-size (mm)

Fig. 4. (a) Photograph of one of polluted sub-areas at Cuno site; all the cobbles and boulders are coated with fuel (black), two white spheres were placed in the scenario, (b) Image analysis performed with the Split Program, (c) Grain size distribution corresponding to initial photograph.

fuel r es idues . T h e fa te of h y d r o c a r b o n s revea led very s imilar p a t t e r n s t o t h o s e a t C u n o site (F ig . 5, r a p i d d e p l e t i o n in t h e first m o n t h s a n d t h e n a s low­ing d o w n of t he p rocess ) . I n a d d i t i o n , we focussed o n a 1 yea r c o m p a r i s o n b e t w e e n pa rce l s t h a t h a d u n d e r g o n e n a t u r a l a t t e n u a t i o n vs. t h o s e for w h i c h S200 w a s app l i ed . Before field a p p l i c a t i o n of t h e p r o d u c t , l a b o r a t o r y t es t ing w a s p e r f o r m e d w h i c h con f i rmed t h a t S200 w a s a p p r o p r i a t e for use in acce le ra t ing h y d r o c a r b o n b i o d e g r a d a t i o n , as

recen t ly r e p o r t e d (D iez et al . , 2005 ; G a l l e g o et al . , 2005) . A t t h e field si te, fuel l o a d d e t e r m i n e d t h e o p t i m u m a m o u n t of fertil iser for b i o s t i m u l a t i o n ; hence , c a l cu l a t i ons w e r e m a d e as exp l a ined a b o v e ( a n e x a m p l e of i m a g e ana lys i s a n d g r a i n size de te r ­m i n a t i o n is s h o w n in F ig . 4) . R e s u l t s r evea led a t h i cknes s of fuel c o a t i n g s r a n g i n g f r o m a b o u t 0.1 m m to 2 c m , so a n a p p r o x i m a t e fuel l o a d of 3 . 5 k g / m 2 a t t h e M o r e i r a site (S200 w a s d o s e d in a c c o r d a n c e w i t h these d a t a -see a b o v e ) .

C 1 7 + C 1 8 / h o p a n e C30+C31 + C 3 2 + C 3 3 / h o p a n e

-Na tu ra l at tenuat ion

Biost imulat ion with S-200

May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04

P r + P h / h o p a n e

Natural at tenuat ion

Biost imulat ion wi th S-200

r-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04

S C 2 - D B T / h o p a n e

0 Natural at tenuat ion

— • — Biost imulat ion with S-200

r -

100 -,

90 •

80 •

70 • c o 60 • o a. 50 • o D 40 • 5?

30 •

20 •

10 •

o A

- N a t u r a l at tenuat ion

Biost imulat ion wi th S-200

Mar-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04

£ ( C 1 - A P h + C 2 - A P h ) / h o p a n e

- N a t u r a l a t tenuat ion

Biost imulat ion wi th S-200

Mar-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04

P y r e n e / h o p a n e

100 -,

90 •

80 -

c 70 -o

60 • "5 a. o

50 -D 40 -

30 •

20 •

10 -

0 <

- N a t u r a l at tenuat ion

Biost imulat ion wi th S-200

0 ^ , , , , , , , 0 ^ , , , , , , ,

Mar-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04 Mar-03 May-03 Jul-03 Sep-03 Nov-03 Jan-04 Mar-04 May-04

Fig. 5. Depletion indices measured at Moreira site (see text) during a 1 year sampling period. N.B. in June 2003, half of the parcels studied at the beach were biostimulated with S 200; application was repeated in July and August 2003. Results are averages of duplicate samples taken at the S200-treated and untreated sub-areas, respectively. Errors are within % in Table 1. (Pr: pristane, Ph: phytane; Ci-APh: methyl anthracenes and phenanthrenes; C2-APh: dimethyl anthracenes and phenanthrenes; C 2-DBT: dimethyldibenzothiophenes; hopane: 17a(H), 21P(H), hopane).

T h e resu l t s (F ig . 5) r evea led t h a t t h e p r o d u c t ach ieved a n a d d i t i o n a l h y d r o c a r b o n d e p l e t i o n r a n g ­ing f r o m 10% u p to 30%> d e p e n d i n g o n t h e fuel c o m ­p o u n d s , in c o m p a r i s o n w i t h n a t u r a l a t t e n u a t i o n r a t i o s ; t h e on ly e x c e p t i o n w a s w a x e s ( long c h a i n n-a lkanes ) t h a t p r o b a b l y in ter fere in s o m e w a y w i t h t he l o n g c h a i n oleic c o m p o u n d s in t he fertilizer. F u r t h e r m o r e , it is in t e re s t ing t o p o i n t o u t t h a t m i c r o b i a l c o u n t s in T S B m e d i u m a v e r a g e d 1 0 6

C F U / g fuel before S200 a p p l i c a t i o n a n d d id n o t inc rease significatively d u r i n g t h e fo l lowing m o n t h s ; h o w e v e r , t h e p r o p o r t i o n of t h o s e m i c r o o r g a n i s m s c a p a b l e of g r o w i n g in t h e selective D S m e d i u m w a s d o u b l e d f r o m 20%> to 40%>, i n d i c a t i n g a n enr ich­m e n t in t he h y d r o c a r b o n d e g r a d i n g c o m m u n i t y , w h i c h likely i m p r o v e d d e p l e t i o n indices . I n fact , t he efficiency of o leoph i l i c fert i l izers is b a s e d o n the i r r e s i s t ance t o phys ica l w a v e a c t i o n a n d the i r h y d r o p h o b i c a n d emuls i fy ing p r o p e r t i e s t h a t facili­t a t e s m i c r o b i a l access t o h y d r o c a r b o n s ( S a n t a s et al . , 1999; M u r a d o et al . , 2004) , t h e r e b y p r o m o t i n g g r o w t h of h y d r o c a r b o n - d e g r a d i n g b a c t e r i a . H o w ­ever , a t t he sites s t ud i ed in th i s w o r k , a n d desp i t e ini t ia l successful r e su l t s , t h e p r o d u c t effect d id n o t pers is t t h r o u g h t h e fo l lowing w i n t e r a n d sp r ing , so n o n - c o s t effective successive a p p l i c a t i o n s w o u l d h a v e b e e n n e e d e d .

T h e overa l l r e su l t s of these e x p e r i m e n t s p r o v e d t h a t N a n d P c o n t e n t s a r e n o t t h e on ly l imi t ing p a r a m e t e r s t h a t i m p e d e d b i o d e g r a d a t i o n . C o n ­versely, t w o fac to r s s t rong ly h i n d e r e d t h e p r o g r e s s of b i o d e g r a d a t i o n ; first, t h e a b o v e m e n t i o n e d h o s ­tile m i c r o e n v i r o n m e n t s of fue l -coa ted s h o r e r o c k s , c o m b i n e d w i t h t h e fuel t h i c k n e s s a n d , s econd , t h e

h i g h c o n t e n t of h e a v y - a lbe i t n o n - t o x i c - oil f rac­t i o n s such as res ins a n d a s p h a l t e n e s (especial ly if we cons ide r t h e re la t ive inc rease in these f r ac t ions after t h e r a p i d ini t ia l d e p l e t i o n in l i g h t - m e d i u m ali-p h a t i c s a n d a r o m a t i c s ) . D e t a i l e d eco tox ico log ica l s tud ies s h o u l d e v a l u a t e w h e t h e r t h e p r o d u c t r e m a i n ­ing a l o n g t he shore l ines p o s e s a n e n v i r o n m e n t a l r i sk o r c a n b e c o n s i d e r e d a n ine r t b i t u m i n o u s r e s idue .

3.4. Enhanced natural attenuation by fresh water

D u r i n g t h e first m o n t h s of t he s t u d y r e p o r t e d he re , fuel layers w e r e visibly t h i n n e r a n d t u r n e d b r o w n i s h o r w e r e even c o v e r e d w i t h g reen i sh m a t s in a r e a s w h e r e fresh w a t e r f r o m local s t r e a m s f lowed t h r o u g h t h e p o l l u t e d s h o r e r o c k s . A t first s ight , t h e phys ica l a n d ch emi ca l effects of th i s w a t e r a p p e a r e d t o b e r e s p o n s i b l e for t he c h a n g e s o b s e r v e d in fuel s t r u c t u r e a n d c o l o u r . N e v e r t h e l e s s , fu r the r ana lys i s a t these " h o t s p o t s " r evea led b o t h d e p l e t i o n in b i o ­d e g r a d a b l e h y d r o c a r b o n s (F ig . 6) a n d m i c r o b i a l act ivi t ies p r o m o t i n g b i o d e g r a d a t i o n (F ig . 7) . T h e m i c r o b i a l s t u d y revea led a c o n s i d e r a b l e inc rease in b i o m a s s c o m p a r e d t o t h a t o b t a i n e d f r o m p o l l u t e d s h o r e r o c k s n o t affected by fresh w a t e r flow ( 1 0 7 ¬ 1 0 8 C F U / g vs . 1 0 5 C F U / g fuel). S u b s e q u e n t s a m ­p l ing a n d selective e n r i c h m e n t of cu l tu re s (see M a t e ­r ia ls a n d m e t h o d s ) m a d e it poss ib le t o col lect > 1 0 0 bac t e r i a l a n d a p p r o x i m a t e l y 30 fungal species f r o m the fuel s a m p l e s a t different s tages of b i o d e g r a d a ­t i o n [these m i c r o o r g a n i s m s a r e c u r r e n t l y be ing g r o u p e d a n d a n a l y s e d us ing r e s t r i c t ion f r a g m e n t l eng th p o l y m o r p h i s m ( R F L P ) a n d 16S r D N A s eq u en c i n g m e t h o d s ] .

O Normal shore rocks

• Shore rocks affected by fresh water

0.00 1.00 2 .00 3 .00 1 0 0 5 .00 6.00 7.00

C17+C18/hopane

3.00 9 .00 10.00 11.00

1.70

1.60

1.50

1.40

1.30

1.20

1.10

1.00

0.90

0.80

o o

0 5 .00 6.00

Pr+Ph/hopane

Fig. 6. Representative depletion ratios of fuel samples taken at shore rocks located in the stream flow and others taken at areas close to the former, but without the influence of fresh water. (Pr: pristane, Ph: phytane; Cr-APh: methyl anthracenes and phenanthrenes; C2-APh: dimethyl anthracenes and phenanthrenes; hopane: 17a(H), 21fS(H), hopane).

Fig. 7. (a) Phase contrast (lOOOx) image showing bacterial colonization of fuel hydrocarbon aggregates while growing in selective enrichment experiments in liquid synthetic medium with Prestige fuel oil as sole carbon source, (b) Phase contrast microscopy (lOOOx) of a sample of a microbial mat at Cuno site. At least three different cyanobacteria can be distinguished (arrows), one of which is filamentous (Hormogoneae group), (c) Confocal laser scanning microscopy (CLSM) of a sample from a polluted area with evident oil degradation activity (see text). Samples were stained with propidium iodide (PI) and SYTO 9. Live bacteria appear in green. Oil masses appear as loose (left) or compact (right) reddish conglomerates. Green filament in the centre is probably an alga, (d) The same field observed under differential interference contrast mode.

B a c t e r i a a p p e a r e d t o b e v iab le a n d act ively d e g r a d i n g the h y d r o c a r b o n m a s s w h e n o b s e r v e d u n d e r a con foca l laser s c a n n i n g m i c r o s c o p e (F ig . 7c a n d d ) . M o r e o v e r , m i c r o b i a l o b s e r v a t i o n s e n a b l e d d e t e c t i o n of m i c r o b i a l c o n s o r t i a cons i s t ing of different species of b a c t e r i a a n d c y a n o b a c t e r i a (F ig . 7b) t h a t a r e l ikely t o h a v e p r o m o t e d fuel deg ­r a d a t i o n . D e t a i l e d e x a m i n a t i o n of c y a n o b a c t e r i a s h o w e d the p re sence of different m o r p h o l o g i e s a n d g r o u p s (Anabaena, Oscillatoria a n d o t h e r s ) .

E v i d e n c e of t he c o o p e r a t i o n of m i x e d cu l tu re s in n a t u r a l e n v i r o n m e n t s for b i o d e g r a d a t i o n h a s b e e n r e p o r t e d ( B o u n c h a n et al . , 2000) . I n th i s c o n t e x t , c o m e t a b o l i c a n d synergis t ic r e a c t i o n s ( A l e x a n d e r , 1999; K n a p p a n d B r o m l e y - C h a l l o n e r , 2003) p r o b a ­bly p l a y a special ro le in t h e d e g r a d a t i o n of h igh ly c o m p l e x m i x t u r e s such as t h e Prestige fuel. A t t h e sites s tud ied , t he p re sence of o r g a n i c s u b s t r a t e s (see Site de sc r i p t i on ) , n a t u r a l fresh w a t e r a n d t h e va r i e ty of c o a s t a l m a c r o b i o t i c life d i rec t ly impl ies t he exis tence of h igh ly va r i ed m i c r o b i a l life, as w a s t he case . A l s o , t h e h i s to r ica l ly f r equen t a r r iva l of

smal l oil spills, t a r ba l l s , a n d o t h e r h y d r o c a r b o n sou rces t o t h e a r e a , led t o t he e s t a b l i s h m e n t of p r e ­v ious ly a d a p t e d m i c r o b i a l p o p u l a t i o n s c a p a b l e of r a p i d l y r e s p o n d i n g t o l a rge spills. I n th i s case , it seems t h a t c y a n o b a c t e r i a , poss ib ly fungi , a n d a s so ­c ia ted a e r o b i c b a c t e r i a a p p e a r t o f o r m a c o n s o r t i u m t h a t fos ters fuel b i o d e g r a d a t i o n ( B e n d e r a n d Phi l ­l ips, 2004) a n d , u n d e r o p t i m a l c o n d i t i o n s , w o u l d b e ex t r eme ly effective, as r evea led b y t h e inc reas ing b i o d e g r a d a t i o n effects visible in t h e c h r o m a t o g r a m s (F ig . 8) .

I n fact , t h e r e is n o d o u b t t h a t c y a n o b a c t e r i a p l a y a n i m p o r t a n t ro le in these m i c r o b i a l m a t s b y e s t a b ­l ishing o x y g e n g r a d i e n t s a n d / o r s u p p l y i n g n u t r i e n t s for h e t e r o t r o p h i c b a c t e r i a ( A l - H a s a n et al . , 1998; C o h e n , 2002) . N e v e r t h e l e s s , g iven t h a t t h e r e is n o sci­entific ce r t a in ty t h a t p o i n t s t o a d i rec t ro le of c y a n o ­b a c t e r i a in d e g r a d a t i o n p a t h w a y s ( A b e d a n d K o s t e r , 2005) , w e sugges t t h a t , as p rev ious ly p r o ­p o s e d ( C o h e n , 2002) , h e t e r o t r o p h i c a e r o b i c b a c t e r i a a re r e s p o n s i b l e for t h e h y d r o c a r b o n d e g r a d a t i o n a t t h e sites. I n th i s c o n t e x t , t h e oi l - insensi t ive

Ph

Pr

15.00 2 0 . 0 0 2 5 . 0 0 30 .00 35 .00 4 0 . 0 0 4 5 . 0 0

Ik

15.00 2 0 . 0 0 2 5 . 0 0 30 .00 35 .00 4 0 . 0 0 4 5 . 0 0

Ph Pr

ill1

Hopanes

2 4 00 26 00 2 8 00 30 00 32 00 34 00 36 00 38 00 4 0 00 4 2 00 4 4 00 4 6 00 4 8 00

Hopanes

C,-C, APh

uj V 2 4 00 2 6 00 2 8 00 30 00 32 00 34 00 36 00 3 8 00 4 0 00 4 2 00 44 00 4 6 00 4 8 00

Hopanes

C,-C, APh

2 4 00 2 6 00 2 8 00 30 00 32 00 34 00 36 00 3 8 00 4 0 00 4 2 00 44 00 4 6 00 4 8 00

Fig. 8. SIM chromatograms (m/z 57, m/z 191 right) after sampling a fuel-polluted boulder partially submerged in fresh water at Cuno site, (a) corresponds to the fuel scraped from upper part of the rock (never submerged) where the fuel was black; (b) is result of GC-MS analysis of fuel scraped from the intermediate part where it was intermittently submerged in fresh water and appeared brownish; (c) was obtained after analysis of fuel that was always submerged in fresh water and covered by a microbial mat. (Pr: pristane; Ph: phytane; C[-C 2

APh: methyl and dimethyl anthracenes and phenanthrenes).

c y a n o b a c t e r i a w o u l d p r o v i d e t h e m o l e c u l a r o x y g e n for a e r o b i c m e t a b o l i s m , as well as c o o p e r a t i n g in t he phys ica l d e s e g r e g a t i o n of t h e fuel, w h e r e a s a e r o ­b ic b a c t e r i a w o u l d m a k e use of typ ica l m e c h a n i s m s , i.e. b i o s u r f a c t a n t a n d bioemuls i f ier p r o d u c t i o n (F ig . 7a ) , t o faci l i ta te the i r o w n access t o h y d r o c a r ­b o n s ( G a l l e g o e t al . , 2 0 0 1 ; R o n a n d R o s e n b e r g , 2001) .

A s e x p l a i n e d a b o v e , th i s m i c r o b i a l p o t e n t i a l is e n h a n c e d on ly in specific a r ea s w h e r e h u m i d i t y , n u t r i e n t levels a n d a va r i e ty of m i c r o o r g a n i s m s a r e m a i n t a i n e d by t h e gen t le flow of fresh w a t e r . M o r e i m p o r t a n t l y , th i s f r e shwa te r flow w e a k e n s fuel a d h e ­s ion a n d so increases b ioava i l ab i l i ty , d r a m a t i c a l l y e n h a n c i n g b i o d e g r a d a t i o n . O n t h e o t h e r h a n d , w h e r e v e r a flow of fresh w a t e r is ab sen t , t h e i n h o s ­p i t a b l e m i c r o e n v i r o n m e n t o n t h e s h o r e r o c k sur­faces, c o m b i n e d w i t h t h e p h y s i c o c h e m i c a l

cha rac te r i s t i c s of t h e fuel (heavy a n d v i scous , a n d h i g h res in a n d a s p h a l t e n e c o n t e n t s ) all b u t i m p e d e m i c r o b i a l ac t iv i ty . T o o v e r c o m e th i s p r o b l e m , n e w b i o r e m e d i a t i o n eng inee r ing s t ra teg ies b a s e d o n irr i­g a t i o n of sho re r o c k s a r e n e e d e d ( G a l l e g o et al . , u n p u b l i s h e d resu l t s ) .

4. Conclusions

1. T h e fa te of h y d r o c a r b o n s a t shore l ines affected b y oil spills is h igh ly c o n d i t i o n e d b y t h e signifi­c a n t h e t e r o g e n e i t y of t h e oil l o a d , w e a t h e r i n g p roces ses a n d m i c r o b i a l ac t iv i ty , a m o n g o t h e r f ac to r s . T h e r e f o r e , r i g o r o u s s a m p l i n g p r o t o c o l s s h o u l d b e i m p l e m e n t e d t o quan t i fy fuel l o a d s o n s h o r e r o c k s a n d g e o c h e m i c a l c h a n g e s s h o u l d b e sys temat ica l ly m o n i t o r e d by us ing re l iab le ana ly t i ca l p r o c e d u r e s . I n th i s w o r k , nove l m e t h -

o d s of i m a g e ana lys i s h a v e b e e n d e v e l o p e d t o m e e t t h e first objec t ive a n d will b e useful in fu tu re c o m p a r a b l e s tud ies . I n a d d i t i o n , t h e d e m ­o n s t r a t e d usefulness of h o p a n e b i o m a r k e r s , espe­cially 1 7 a ( H ) , 2 1 p ( H ) h o p a n e , e n a b l e d u s t o effectively m o n i t o r t h e e v o l u t i o n of different h y d r o c a r b o n famil ies .

2 . T h e Prestige fuel oil t h a t r e a c h e d t h e S p a n i s h c o a s t s h a d chemica l cha rac t e r i s t i c s (h igh p r o p o r ­t i o n of a s p h a l t e n e s a n d resins) t h a t h i n d e r e d r a p i d w e a t h e r i n g , specifically b i o d e g r a d a t i o n , sugges t ing t h a t t h e b ioava i l ab i l i t y of h e a v y frac­t i o n s w a s ex t r eme ly l ow at m o s t of t he si tes. M o r e t h a n t w o yea r s after t h e spill , t h e sites w h e r e n o r e m e d i a t i o n t r e a t m e n t w a s p e r f o r m e d still m a i n t a i n > 5 0 % of t h e ini t ia l a m o u n t of a r o ­m a t i c c o m p o u n d s ; h o w e v e r , l ight a n d m e d i u m n-a l k a n e s w e r e a l m o s t t o t a l ly d e g r a d e d in t h e i m m e d i a t e m o n t h s fo l lowing t h e spill .

3 . T h e h y d r o c a r b o n d e p l e t i o n w a s e n h a n c e d a t s u b -a r e a s w h e r e t h e o leoph i l i c ferti l izer S200 w a s app l i ed . M o r e o v e r , m i c r o b i o l o g i c a l ana lys i s r evea led a n i m p o r t a n t g r o w t h of h y d r o c a r b o n d e g r a d i n g p o p u l a t i o n s w h e r e v e r S200 w a s app l i ed . A s a c o n s e q u e n c e , th i s p r o d u c t c a n b e c o n s i d e r e d a r e a s o n a b l e o p t i o n for b i o r e m e d i a -t i o n of m a r i n e oil spills.

4 . H y d r o c a r b o n - d e g r a d i n g m i c r o o r g a n i s m s w e r e wide ly d i s t r i b u t e d a t t he p o l l u t e d si tes, r evea l ing r a p i d a d a p t a t i o n t o t h e p re sence of l a rge a m o u n t s of fuel a n d ru l i ng o u t a n y necess i ty for a p p l y i n g b i o a u g m e n t a t i o n . H i g h l y c o m p l e x c o n s o r t i a of e u k a r y o t i c a n d p r o k a r y o t i c m i c r o b e s ( c y a n o b a c ­te r ia , fungi a n d bac t e r i a ) c o n c e i v a b l y e n a b l e d d e g r a d a t i o n of fuel t o t a k e p lace w h e n p a r a m e t e r s such as h u m i d i t y , d i s so lved o x y g e n a n d n u t r i e n t ava i lab i l i ty w e r e o p t i m a l a n d fuel a d h e s i o n w a s phys ica l ly w e a k e n e d . H o w e v e r , these c o n d i t i o n s w e r e p r e s e n t a n d so on ly f a v o u r a b l e in ce r t a in , smal l a r ea s of t h e c o n t a m i n a t e d sites w h e r e fresh w a t e r f lowed t h r o u g h t h e s h o r e r o c k s . T h i s c o n ­c lus ion sugges ts fu r the r nove l b i o r e m e d i a t i o n a p p r o a c h e s b a s e d o n i r r i ga t i on w i t h fresh w a t e r f lowing in to t h e c o n t a m i n a t e d sites.

Acknowledgements

T h i s w o r k rece ived a g r a n t f r o m t h e " C o n s e j o S u p e r i o r de Co leg ios d e I n g e n i e r o s d e M i n a s d e E s p a n a " . T h e a u t h o r s w o u l d l ike t o express the i r a p p r e c i a t i o n t o P ro fe s so r T o m a s D i a z for

a s s i s t ance in c y a n o b a c t e r i a c h a r a c t e r i z a t i o n , t o Pr isc i l la C h a s e for l inguis t ic r ev i s ion of t h e m a n u ­scr ip t a n d t o t w o a n o n y m o u s rev iewers for helpful c o m m e n t s .

Guest Associate Editors—F.J. Gonzalez-Vi la a n d J . A . Gonzalez-Perez

References

Abed, R.M., Koster, J., 2005. The direct role of aerobic heterotrophic bacteria associated with cyanobacteria in the degradation of oil compounds. International Biodeterioration & Biodegradation 55, 29-37.

Alexander, M., 1999. Biodegradation and Bioremediation, 2nd ed. Academic Press, London.

Al-Hasan, R.H., Al-Bader, D.A., Sorkhoh, N.A., Radwan, S.S., 1998. Evidence for «-alkane consumption and oxidation by filamentous cyanobacteria from oil-contaminated coasts of the Arabian Gulf. Marine Biology 130, 521-527.

Alzaga, R., Montuori, P., Ortiz, L., Bayona, J.M., Albaiges, J., 2004. Fast solid-phase extraction-gas chromatography-mass spectrometry procedure for oil fingerprinting - Application to the Prestige oil spill. Journal of Chromatography A 1025, 133-138.

Bender, J., Phillips, P., 2004. Microbial mats for multiple applications in aquaculture and bioremediation. Bioresource Technology 94, 229-238.

Benoit, Y., Haeseler, F., 2004. Weathering of tanker Prestige and Erika fuel oils no. 6 in Marine environments. In: Proceedings of Interspill conference 2004, Trondheim, Norway, Section 6, p. 460.

Bost, F.D., Frontera-Suau, R., McDonald, T.J., Peters, K.E., Morris, P.J., 2001. Aerobic biodegradation of hopanes and norhopanes in Venezuelan crude oils. Organic Geochemistry 32, 105-114.

Bouchez, M., Blanchet, D., Bardin, V., Haeseler, F., Vandecas-teele, J.P., 2000. Efficiency of defined strains and of soil consortia in the biodegradation of polycyclic aromatic hydrocarbon (PAH) mixtures. Biodegradation 10, 429^35.

Bounchan, S., Britz, M.L., Stanley, G.A., 2000. Degradation and mineralization of high-molecular-weight polycyclic aromatic hydrocarbons by defined fungal-bacterial cocultures. Applied Environmental Microbiology 66, 1007-1019.

Bragg, J.R., Prince, R.C., Harner, J., Atlas, R.M., 1994. Effectiveness of bioremediation for the Exxon Valdez oil spill. Nature 368, 413^18.

Cohen, Y., 2002. Bioremediation of oil by marine microbial mats. International Microbiology 5, 189-193.

Daling, P.S., Strom, T., 1999. Weathering of oils at sea: Model/ Field Data Comparisons. Spill Science & Technology 5, 63¬ 74.

Diez, S., Sabate, J., Vinas, M., Bayona, J.M., Solanas, A.M., Albaiges, J., 2005. The Prestige oil spill. I. Biodegradation of a heavy fuel oil under simulated conditions. Environmental Toxicology and Chemistry 24, 2203-2217.

Fafet, A., Da Silva, M., Haeseler, F., 2004. Analytical charac­terization and comparison of Prestige and Erika's fuel oil n°6. In: Proceedings of the Interspill conference 2004, Trondheim, Norway, Section 6, p. 462.

Gallego, J.R., Loredo, J., Llamas, J.F., Vazquez, F., Sanchez, J., 2001. Bioremediation of diesel-contaminated soils: Evaluation of potential in situ techniques by study of bacterial degrada­tion. Biodegradation 12, 325-335.

Gallego, J.R., Menendez-Vega, D., Gonzalez-Rojas, E., Sanchez, J., Garcfa-Martfnez, M.J., Llamas, J.F., 2005. Oleophylic fertilizers and bioremediation: a new perspective. Book of abstracts, Biomicroworld. In: 1st Conference on Environ­mental, Industrial and Applied Microbiology. Badajoz (Spain), pp. 714-715.

Ghazali, F.M., Abdul Rahman, R., Bakar Salleh, A., Basri, M., 2004. Biodegradation of hydrocarbons in soil by microbial consortium. International Biodeterioration & Biodegradation 54, 61-67.

Head, I.M., Swannell, R.P., 1999. Bioremediation of petroleum hydrocarbon contaminants in marine habitats. Current Opinion Biotechnology 10, 234-239.

Hostettler, F.D., Kvenvolden, K.A., 1994. Geochemical changes in crude oil spilled from the Exxon-Valdez supertanker in Price William Sound, Alaska. Organic Geochemistry 21, 927¬ 936.

Hurst, C.J., 2002. Neighborhoods and community involvement: no microbe is an island. In: Hurst, C.J. (Ed.), Manual of Environmental Microbiology, Second ed. ASM Press, Wash­ington, pp. 6-18.

Kanaly, R.A., Bartha, R., Watanabe, K., Harayama, S., 2000. Rapid mineralization of benzo(a)pyrene by a microbial consortium growing on diesel fuel. Applied Environmental Microbiology 66, 4205-4211.

Knapp, J.S., Bromley-Challoner, K.C., 2003. Recalcitrant organic compounds. In: Duncan, M., Nigel, H. (Eds.), Handbook of Water and Wastewater Microbiology. Aca­demic Press, London, pp. 559-595.

Lee, K., Gauthier, J., Cobanli, S.E., Griffin, M., 1997. Bioaug­mentation and biostimulation: a paradox between laboratory and field results. In: Proceedings of the 1997 International Oil Spill Conference, American Petroleum Institute, Washington, Publication No. 4651, pp. 697-705.

Jezequel, R., Menot, L., Merlin, F.X., Prince, R.C., 2003. Natural clean up of heavy fuel oil on rocks: an in situ experiment. Marine Pollution Bulletin 46, 983-990.

Maki, H., Hirayama, N., Hiwatari, T., Kohata, K., Uchiyama, H., Watanabe, M., et al., 2003. Crude oil bioremediation field experiment in the Sea of Japan. Marine Pollution Bulletin 47, 74-77.

Medina-Bellver, J.I., Marin, P., Delgado, A., Rodriguez-Sanchez, A., Reyes, E., Ramos, J.L., et al., 2005. Evidence for in situ crude oil biodegradation after the Prestige oil spill. Environ­mental Microbiology 7, 773-779.

Mishra, S., Jyot, J., Kuhad, R.C., Lai, B., 2001. Evaluation of inoculum addition to stimulate in situ Bioremediation of Oily-Sludge-Contaminated soil. Applied Environmental Microbi­ology 67, 1675-1681.

Moldestadt, M., Daling, P., Leirwik, F. The Prestige oil -properties and weathering at sea., 2004. In: Proceedings of Interspill conference 2004, Trondheim, Norway, Section 6, p. 461.

Murado, M.A., Miron, J., Gonzalez, M., Vazquez, J., Cabo, M., Pintado, J., 2004. Prestige oil spill. Results of bioremediation assays on supra-tidal rocks of Salvora Island (Galice, Spain). In: Proceedings of Interspill conference 2004, Trondheim, Norway, Section 1, p. 480.

Oh, Y.S., Sim, D.S., Kim, S.J., 2001. Effects of nutrients on crude oil biodegradation in the upper tidal zone. Marine Pollution Bulletin 42, 1367-1372.

Page, C.A., Bonner, J.S., McDonald, T.J., Autenrieth, R.L., 2002. Behaviour of a chemically dispersed oil in a wetland environment. Water Research 36, 3821-3833.

Peters, K.E., Moldowan, J.M., 2004. The Biomarker Guide: Interpreting Molecular Fossils in Petroleum and Ancient Sediments, second ed. Prentice Hall, New Jersey.

Pineda-Flores, G., Boll-Argiiello, G., Lira-Galeana, C , Mesta-Howard, A.M., 2004. A microbial consortium isolated from crude oil that grows in asphaltenes as only source of carbon and energy. Biodegradation 15, 145-151.

Pollard, S.J., Whittaker, M., Risden, G.C., 1999. The fate of heavy oil wastes in soil microcosms I: a performance assessment of biotransformation indexes. The Science of the Total Environment 226, 1-22.

Prince, R.C., Elmendorf, D.L., Lute, J.R., Hsu, C.S., Haith, C.E., Senius, G.J., et al., 1994. 17oi(H), 21|3(H)-hopane as a conserved internal biomarker for estimating the biodegrada­tion of crude oil. Environmental Science & Technology 28, 142-145.

Pritchard, P.H., Mueller, J.G., Rogers, J.C., Kremer, F.V., Glaser, J.A., 1992. Oil spill bioremediation: experiences, lessons and results from the Exxon Valdez oil spill in Alaska. Biodegradation 3, 315-335.

Ron, E.Z., Rosenberg, E., 2001. Natural roles of bio surfactants. Environmental Microbiology 3, 229-236.

Santas, R., Korda, A., Tenente, A., Buchholz, K., Santas, P.H., 1999. Mesocosm assays of oil spill bioremediation with oleophylic fertilizers: Inipol, Fl or both? Marine Pollution Bulletin 38, 44-48.

Speight, J.G., 2001. Handbook of Petroleum Analysis. John Wiley and Sons Inc, New York.

Sasaki, T., Maki, H., Ishihara, M., Harayama, S., 1998. Vanadium as an internal marker to evaluate microbial degradation of crude oil. Environmental Science & Technol­ogy 32, 3618-3621.

Venosa, A.D., Zhu, X., 2003. Biodegradation of crude oil contaminating marine shorelines and freshwater wetlands. Spill Science & Technology Bulletin 8, 163-178.

Wackett, L.P., Hershberger, C D . , 2001. Biocatalysis and bio­degradation: Microbial transformation of organic com­pounds. ASM Press, Washington.

Wang, Z., Fingas, M.F., 2003. Development of oil hydrocarbon fingerprinting and identification techniques. Marine Pollution Bulletin 47, 423^152.

Whittaker, M., Pollard, S.J., Risden, G., 1999. The fate of heavy oil wastes in soil microcosms II: a performance assessment of source correlation indexes. The Science of the Total Environ­ment 226, 23-24.

Xu, R., Obbard, J.P., 2004. Biodegradation of polycyclic aromatic hydrocarbons in oil-contaminated beach sediments treated with nutrient amendments. Journal of Environmental Quality 33, 861-867.

Zhu, X., Venosa, A., Suidam, M., Lee, K., 2001. Guidelines for the bioremediation of marine shorelines and freshwater wetlands. U.S. Environmental Protection Agency. Office of Research and Development National Risk Management Research Laboratory. Land Remediation and Pollution Control Division, Cincinnati, USA (Available at www.epa. gov/oilspill/pdfs/bioremed.pdf).